Abstract

Damaging inflammation arising from autoimmune pathology and septic responses results in severe cases of disease. In both instances, anti-inflammatory compounds are used to limit the excessive or deregulated cytokine responses. We used a model of robust T cell stimulation to identify new proteins involved in triggering a cytokine storm. A comparative proteomic mining approach revealed the differential mapping of Raf kinase inhibitory protein after T cell recall in vivo. Treatment with locostatin, an Raf kinase inhibitory protein inhibitor, induced T cell anergy by blocking cytokine production after Ag recall. This was associated with a reduction in Erk phosphorylation. Importantly, in vivo treatment with locostatin profoundly inhibited TNF-alpha production upon triggering the Ag-specific T cells. This effect was not limited to a murine model because locostatin efficiently inhibited cytokine secretion by human lymphocytes. Therefore, locostatin should be a useful therapeutic to control inflammation, sepsis, and autoimmune diseases.

Exacerbated cytokine response after recall of adoptively transferred OT-I cells. A, C57BL/6 mice adoptively transferred with 150,000 OT-I cells were immunized with OVA, poly I:C, and anti-4-1BB (CD137) Ab and rested for 6 days. Spleen cells were harvested before (No Recall) and 10–90 min after in vivo recall with 100 μg SIINFEKL peptide. Cells were fixed, permeabilized, and stained for IFN-γ and for the congenic marker CD45.1 (peptide-specific CD8+ T cells). The percentage of OT-I cells (CD45.1+) producing IFN-γ is indicated. B, The mean percentage ± SEM of OT-I cells producing IFN-γ from three independent biological replicates is shown. C, Sera were obtained and quantified for TNF-α presence by ELISA and shown as individual scatter plots. Because the data were not normally distributed, a Mann-Whitney U test was used and the results showed a p < 0.05 compared with 10 min or no recall.

Differential proteomic fingerprint of Resting vs Recall cells. Ai, C57BL/6 mice transferred with OT-I cells were immunized as described in legend of and recalled for 3 h after i.p injection with 100 μg SIINFEKL (Recall), or left untouched (Resting). Splenocytes and lymph node cells were harvested, lysed, and applied to the PF 2D platform. Aii, The lysate was processed on a Beckman Coulter ProteomeLab PF 2D platform. The chromatofocusing was performed as linear gradient from ph 8.0 to 4.0. Fractions were collected in 0.3 pH intervals, automatically reinjected for a second dimension on C18 column at 50°C. Two-dimensional protein expression maps displaying protein isoelectric point (pI) vs protein hydrophobicity were generated by Proteo-View/DeltaVue software package. B, A comparative two-dimensional map representative of one out four in vivo experiments is shown (left). Each experiment identified similar chromatographic profiles we call signatures as well as differential ones we call finger-prints. In every one of the four experiments (Right, no. 1 to 4) we found, at the same precise coordinates on the proteomic maps, a reproducible fingerprint showing a peak present in the recall and absent (or smaller) in the resting sample.

Identification of RKIP as a fingerprint in recall cells. A, One second dimension fraction from the recall sample and its corresponding equivalent from the no recall sample were lyophilized and resolved by 4–15% SDS-PAGE. A 22 kDa band, detected by a protein-specific fluorescent dye was cut, digested by trypsin, sequenced by MALDI-TOF. Peptide sequences were searched against using the NCBInr database version 20060804 using the Proteometrics Software Suite and the Profound Search Algorithm. B, RKIP expression was assessed by immunobloting of the original sample (left), the cell lysate immediately before loading on the PF 2D (middle), and the kinetics of an in vivo recall response (right).

Locostatin, a RKIP-specific compound, induces T cell anergy in vitro. A, Splenocytes and LN cells 6 days after immunization as described in legend of were restimulated in vitro for 5 h with SIINFEKL in the absence or presence of locostatin. Cells were fixed, permeabilized, and stained for IFN-γ, TNF-α and the congenic marker CD45.1. The percentage CD45.1+-gated cells producing IFN-γ and TNF-α is indicated. B, The percentage ± SEM of OT-I cells (CD45.1+) producing IFN-γ and TNF-α from five independent experiments (n = 16) is shown for four incremental doses of locostatin: 0 μM, 6.7 μM, 20 μM, and 60 μM. C, Inhibition of cytokine secretion was tested by adding the 60 μM of locostatin at 0, 10, 60, and 180 min during a 300 min SIINFEKL restimulation. The percentage CD45.1+-gated cells producing IFN-γ and TNF-α is representative of three independent experiments (n = 14).

Locostatin inhibits the Erk pathway. A, Splenocytes and LN cells 6 days after immunization as described in legend of were tested for activation of the Erk pathway. Representative Western blots from five independent biological replicates are shown. Cells were preincubated with 60 μM locostatin for 1 h followed by restimulation with/without SIINFEKL for 1 h. Cell lysates were resolved by 4–15% gradient SDS-PAGE under reducing and denaturing conditions, transferred to nitrocellulose membrane and probed with the indicated primary Ab as described in the Materials and Methods section. B, In three separate experiments, splenocytes and LN cells from naive Rag−/− OT-I mouse were treated with locostatin and analyzed as described in A. C, OT-I cells, purified from splenocytes and LN cells 6 days after immunization were plated on plate-bound CD3 for 10 min in presence or absence of 60 μM locostatin. Cell lysates were obtained and processed as described in A. D, Splenocytes and LN cells 6 days after immunization, in one experiment (n = 2), were preincubated with DMSO or 100 μM locostatin (RKIP inhibitor), 100 μM disulfiram (aldehyde dehydrogenase 1A1 and NFκB inhibitor), 100 μM ethacrynic acid (glutathione S-transferase inhibitor), 100 μM Z-Pro-Pro-CHO (prolyl oligopeptidase inhibitor) for 20 min. followed by restimulation with SIINFEKL for 1 h. Cell lysates were obtained and processed as described in A.

Locostatin inhibit exacerbated TNF-α secretion. A, Mice immunized as described in legend of were injected i.p. with 100 μg of locostatin or vehicle 15 min before i.p. injection with 100 μg of SIINFEKL. A summary of three independent experiments is shown. The mice were bled 1 h later; sera were tested for TNF-α presence by ELISA (pg/ml) and shown as individual scatter plots. B, In a complementary experiment, human anti-flu CTL were stimulated for 16 h with increasing doses of cognate peptide in the presence of locostatin. TNF-α and IFN-γ release was measured by ELISA. C, Human PBMC were stimulated for 3 h with increasing doses of LPS in the presence of locostatin. TNF-α and IFN-γ release was measured by ELISA.